Dataset Information

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.

ABSTRACT: Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).

SUBMITTER: Mann G

PROVIDER: S-EPMC9369648 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Publications

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.

Mann Guy G Sulkshane Prasad P Sadhu Pradeep P Ziv Tamar T Glickman Michael H MH Brik Ashraf A

Molecules (Basel, Switzerland) 20220729 15

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitorin ...[more]

PMID: 35956818

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Project description:Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65).

Dataset Information

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.

Publications

Antibody for Serine 65 Phosphorylated Ubiquitin Identifies PLK1-Mediated Phosphorylation of Mitotic Proteins and APC1.

Similar Datasets

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